A large number of the human genetic diseases result from deficiencies of enzyme activity in metabolic pathways. Some of the enzyme deficiency diseases can be ameliorated by dietary restriction, enzyme replacement, or enzyme manipulation, but this approach has only limited applications. Allotransplantation of tissues from a genetically normal donor into a patient to provide a source of normal enzyme has produced encouraging results in a small number of animal and human patients. However, this approach is limited by the frequent lack of a histocompatible donor, and by the relatively high morbidity and mortality of the procedure even when a matched donor is available. Somatic cell gene therapy has the potential to overcome these limitations by transferring a normal copy of a defective gene into a patient's own cells and returning the corrected cells to the patient's body by autologous transplantation.
The objective of gene therapy is to permanently correct the genetic defect in the target cells. Although showing great promise, gene therapy has encountered a number of difficulties.
The gene involved in the disease must be identified and cloned. Although self-apparent, this has not been accomplished for many genetic diseases. At the present time, the lack of cloned genes precludes the use of many animal models of human genetic disease for gene therapy studies.
A vector system must be developed that is capable of stably transferring the gene into the appropriate target cell. Several methods have been developed to transfer cloned genes into mammalian cells, achieve stable integration into the host cell DNA, and express the transferred gene. The most widely used method for gene therapy studies is the replication-defective retroviral vector system, which can transfer genes into a high percentage of target cells in tissue culture. However, the types of cells and, consequently, the diseases that are candidates for gene therapy using this method are limited by the fact that target cells must undergo mitosis for vector provirus integration to occur.
The transferred gene must be appropriately expressed in the diseased target cells. A major problem encountered in retroviral vector gene transfer into somatic cells, particularly bone marrow cells, has been that the expression of the inserted gene is either abrogated or substantially reduced when the cells containing the transferred gene are returned to the animal host. The failure of transduced hematopoietic cells to maintain expression of a vector gene in vivo may be caused by vector dysfunction, poor survival of the transfected cells, selection against the transfected cells by the host, or low efficiency of transfer into true pluripotent hematopoietic stem cells. Inadequate expression of the transferred gene in vivo may also be a consequence of not including all of the necessary regulatory elements in the vector design. To overcome this obstacle, new vectors have achieved improved expression by using internal promoters to regulate expression, by including certain intronic genomic sequences and, in the double-copy (DC) vectors, by moving the transferred gene outside the retroviral transcription unit. The correctly expressed transferred gene must be able to cure or significantly alter the course of the disease. A crucial test of the feasibility of applying gene therapy methods to human patients will be the demonstration of its effectiveness in altering the pathophysiology of disease in animal models. Progress in gene therapy has been hampered because animal disease models have not yet been discovered or created for most of the human genetic diseases in which the cloned gene is available.
The pluripotent hematopoietic stem cell is a major target for either replacement by transplanted normal allogeneic cells or correction by gene transfer in disorders such as lysosomal storage diseases. The basis for this approach is that lysosomal enzymes can be exported from normal to mutant cells via both receptor-mediated endocytosis and direct intercellular transfer of the enzyme. Bone marrow transplantation studies indicate that circulating blood cells producing normal lysosomal enzymes can export the enzyme to other cell types in sufficient amounts to impede or reverse the storage process in certain tissues. Thus, sustained expression of a vector-transferred lysosomal enzyme gene in autologously transplanted hematopoietic cells, when achievable, may be an effective approach to therapy for some organ systems. However, bone marrow transplantation has not been very effective in lysosomal storage diseases.
Treating the CNS disease will be critical in human patients with disorders of the CNS such as lysosomal storage diseases because many are mentally retarded and may benefit from such treatment. Whether enzymes can be exported to the CNS from donor bone marrow-derived cells has been a difficult problem to address experimentally because most studies have been performed in limited numbers of human patients or outbred domestic animals. Nevertheless, widespread increase of fucosidase enzyme activity in leukocytes, plasma, and visceral tissues was accompanied by a rapid improvement in the peripheral nerve and visceral lesions of fucosidosis and more gradual improvement in the CNS after bone marrow reconstitution in dogs with β-fucosidase deficiency and α-L-iduronidase deficiency. Also, post-transplantation improvements in mental development have been reported in a child with MPS IH and a child with aryl sulfatase-A deficiency. In contrast, in other lysosomal storage diseases little evidence has been found for alteration of the CNS in post-transplant animals. For example, in both Niemann-Pick mice (spm/spm) and MPS VII mice, bone marrow transplantation resulted in increased levels of activity of the missing enzyme and marked improvement in many affected organs such as spleen and liver, but neurological manifestations were not improved, nor was enzyme activity significantly increased in the CNS.
The failure of bone marrow transplantation to affect the CNS may be due to the inability of either hematopoietic cells or exported enzyme to cross the blood-brain barrier. Some experiments suggest that disruption of this barrier may allow some cells or enzyme to enter the CNS. In preliminary studies in MPS VII mice, increasing the dose of radiation to recipient mice resulted in a small increase in the level of β-gus activity in the CNS of long-term chimeras. The infiltration of donor-derived enzymatically competent, foamy macrophages in the CNS of bone marrow transplanted recipients has been shown in “twitcher” mice [Hoogerbrugge et al., “Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation”, Science 239:1035-1038 (1988)]. However, it is not known whether the presence of donor cells in the CNS is due to normal migration of the cells or if normal barriers to such migration have been disrupted by the disease process, in which toxic amounts of psychosine accumulate intercellularly in the CNS as a result of the defect in galactosylceramidase. In addition, even with appropriate allotransplantation, a major complication is fatal sepsis resulting from the immunosuppressive drugs administered to prevent graft rejection.
New approaches are needed to achieve significant alterations in the CNS component of disorders of the CNS. One approach for delivering gene products to the CNS is to engraft genetically altered cells into the brain as a source of a biological molecules [T. Friedmann, “Progress toward human gene therapy”, Science 244:1275-1281 (1989)]. This approach has been used to introduce nerve growth factor (NGF) into rat brains by transplantation of fibroblasts expressing a retroviral vector transferred NGF cDNA. A second approach to delivering gene products to the CNS is using neurotropic viruses as vectors to transfer the gene into CNS cells.
Infection with herpes simplex virus (HSV-1), a neurotropic virus, begins with viral replication in epithelial tissues. After initial replication at the site of infection, HSV-1 establishes latent infection in the nervous system during which no virus can be detected unless reactivation occurs. However, during latency viral DNA can be detected in the CNS of mice and humans [Fraser et al., “Molecular biology of latent HSV-1. In: Human herpes virus infections. II viral glycoproteins and immunobiology”, Raven Press NY 39-55 (1986)]. Latent HSV-1 can be found in more than 80% of humans of which 30% have occasional reactivations in the form of cold sores.
Latency is established in the neuron and the establishment of latency probably depends on the lack of specific factors or specific interactions of the virus with the neuron. Estimates of the proportion of latently infected cells in sensory ganglia are in the range of 0.1-1%, based on reactivation from dissociated cell preparations. It has been suggested that specific viral gene products may be required for the establishment of a latent infection. However, results from experiments using HSV-1 temperature-sensitive mutants (both DNA replication positive and negative) suggest that neither DNA replication nor a productive infection are absolute requirements for establishing a latent infection. Furthermore, recent results suggest that no viral gene product is required for latency.
The acute viral infection is cleared by the immune system but there is little convincing evidence that clearly implicates involvement of the immune system in establishment, maintenance or reactivation of latency.
Using in situ hybridization and Northern blotting, the viral transcripts expressed during latency have been mapped to one small region of the viral genome—the repeat long region. These results in animal models have been repeated with human tissue. The transcripts made during latency have been called latency-associated transcripts (LATs) and their promoter has been mapped to a specific TATAA box within the repeat long region of the viral genome. LAT RNAs do not have an essential role in latency but they may play a role in the efficiency or speed of reactivation of the virus. Data suggest that the lytic and latent viral cycles are separate and that it is the absence of the virion-encoded transactivating factor which causes a latent infection to be established.
The LAT gene product has been seen to accumulate to high levels in the neuronal cells of both latently infected animals and humans for the life of the animal. All other HSV-1 genes are silent during latency. Thus it has been thought that the promoter of the LAT gene could be used to express foreign genes during latency. One group has constructed a virus containing the β-globin gene inserted 26 bases downstream of the TATA box of the LAT promoter and documented expression of the reporter gene in the peripheral nervous system for up to three weeks after establishment of the latency [Dobson et al., “Identification of the latency-associated transcript promoter by expression of rabbit β-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus”, J Virology, vol. 63, No. 9, pp 3844-51 (1989)]. Their analysis of RNA indicated that appropriate splicing, polyadenylation and cytoplasmic transport of the β-globin gene occurred in the spinal ganglia indicating that the neurons of the peripheral nervous system have appropriate transcription and translation machinery. Another group tested a recombinant HSV-1 vector wherein the β-galactosidase gene was placed about 823 base pairs (bp) downstream of the LAT promoter [Ho et al., “Herpes simplex virus latent RNA (LAT) is not required for latent infection in the mouse,” Proc. Natl. Acad. Sci. USA 86:7596-7600 (1989)]. Although the viruses expressed foreign genes during latency in vivo, the documented expression was limited to the peripheral nervous system. Id. Recombinant HSV-1 vectors also have been constructed using the HSV-1 immediated early (IE 4/5) or TK promoters to express the β-galactosidase or HPRT genes [Pallela et al., “Expression of human HPRT in mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector”, Gene, 80:137-44 (1989); Pallela et al., “Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells”, Molecular and Cellular Biology, vol. 8, No. 1 pp 457-60 (1988); Geller, A. I., Breakfield, X. O. “A defective HSV-1 vector expresses Escherichia coli β-galactosidase in cultured peripheral neurons”, Science, 241:1667-69 (23 Sep. 1988); Geller, A. I., Freese, A., “Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli β-galactosidase”, Proc Natl Acad Sci, 87:1149-1153 (1990) and Ho et al., “β-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse,” Virology 167: 279-283 (1988)].
There exists a need for vectors which can deliver foreign genes to, and express them in, the central nervous system preferably on a long term basis in order to modulate biological properties of the central nervous system, such as are found in disease states affecting the central nervous system.